The present invention is directed to a fluoro-thio-containing compound that is capable of specifically inhibiting the enzyme, membrane-bound aminopeptidase P2 (APP2), whose natural substrate is bradykinin. The compound is useful as a pharmaceutical agent because by inhibiting bradykinin degradation, the compound allows endogenous bradykinin to exert its beneficial effects in the body including dilating coronary arteries, providing protective effects in the heart during myocardial ischemia/reperfusion injury, stimulating formation of new blood vessels, improving organ function in chronic heart and renal disease, and improving glucose tolerance and insulin-sensitivity. The present invention is also directed to a pharmaceutical composition comprising the APP2 inhibitor of the present invention and to a method of inhibiting bradykinin degradation in a mammalian patient, particularly a human patient.
More than a million persons in the U.S. have a myocardial infarction (heart attack) each year, resulting in over 500,000 deaths. “Effective treatment of acute myocardial infarction (MI) is based on procedures that promote the return of blood flow to the ischemic zone of the myocardium, i.e., reperfusion therapy.” Ferdinandy et al., “Interaction of Risk Factors, Comorbities, and Comedications with Ischemic/Reperfusion Injury and Cardioprotection by Preconditioning, Postconditioning, and Remote Conditioning,” Pharmacol. Rev. 66:1142-1174 at 1144 (October 2014). “Reperfusion, however, may lead to further irreversible myocardial cell death, termed lethal myocardial reperfusion injury.” Id. “Currently, there is no effective therapy for combined ischemia/reperfusion injury on the market, and routine pharmacologic agents do not salvage the ischemic/reperfused myocardium.” Id. “Therefore, the development of cardioprotective agents to limit the extent of infarcted tissue caused by ischemialreperfusion injury is of great clinical importance.” Id. See also Sivaraman, et al., “Pharmacologic Therapy That Simulates Conditioning for Cardiac Ischemic/Reperfusion Injury,” J. Cardiovascular Pharm. and Therap., 19(1) 83-96 (2014).
One option for treating ischemic/reperfused myocardium after acute myocardial infarction is to increase the heart's concentration of the nonapeptide hormone, bradykinin, at the time when the heart is being reperfused, i.e., when the blood clot in a heart artery is mechanically removed or dissolved. Although restoration of blood flow to the heart reduces heart damage, it is not as effective as it could be because the rapid increase in flow itself causes a different kind of damage called “reperfusion injury.” Bradykinin is known to prevent this kind of injury. However, bradykinin itself is not a good drug because it can seriously lower blood pressure when administered to a patient. Bradykinin is produced in the heart itself during ischemia and reperfusion, but doesn't increase enough to be effective. This is because bradykinin is rapidly degraded by two enzymes: membrane-bound aminopeptidase P2 (APP2) and angiotensin-converting enzyme (ACE) present on blood vessel endothelial cells. Inhibiting APP2 leads to an increase in bradykinin that prevents reperfusion injury. The biochemical mechanisms that cause reperfusion injury and those that prevent it appear to be present in most organs. Therefore, inhibiting APP2 should be protective in most situations where blood flow is stopped for a period of time (ischemia) and then restored (reperfusion). These include, but are not restricted to, ischemia/reperfusion (I/R) of the kidney, brain (stroke), liver, lung, and limb, post-cardiopulmonary bypass surgery, and the whole body I/R such as cardiac arrest and hemorrhagic shock followed by fluid resuscitation. There are currently no approved drugs for preventing reperfusion injury.
The prototype aminopeptidase P inhibitor, apstatin (Formula I), was shown to reduce bradykinin degradation in the isolated perfused rat heart and lung. Apstatin enhanced the blood pressure-lowering effect of intravenously administered bradykinin. In a rat model of
severe hypertension, apstatin, which has no effect by itself, acted synergistically with an ACE inhibitor to reduce blood pressure to normal. [Kitamura et al. “Effects of aminopeptidase P inhibition on kinin-mediated vasodepressor responses,” Am. J. Physiol., 276, H1664-H1671 (1999)] APP2 inhibition with apstatin also exhibited cardioprotective effects; in a heart attack model using an isolated perfused heart, apstatin reduced cardiac damage by 74%. [Ersahin et al., “Cardioprotective effects of the aminopeptidase P inhibitor apstatin: studies on ischemia/reperfusion injury in the isolated rat heart,” J. Cardiovasc. Pharmacol., 34, 604-611 (1999)] It reduced reperfusion-induced ventricular fibrillation by a similar amount. Subsequent studies in other laboratories showed that inhibiting APP2 by administering apstatin substantially reduced myocardial infarct size in intact rats subjected to regional cardiac ischemia. [Wolfrum et al., “Apstatin, a selective inhibitor of aminopeptidase P, reduces myocardial infarct size by a kinin-dependent pathway,” Br. J. Pharmacol., 134, 370-374 (2001); Veeravalli et al., “Infarct size limiting effect of apstatin alone and in combination with enalapril, lisinopril and ramiprilat with experimental myocardial infarction,” Pharmacol. Res., 48, 557-563 (2003).] In the later study, apstatin was effective even when administered during ischemia but just before the start of reperfusion. This indicates that apstatin is specifically protecting against reperfusion injury, since it reached the ischemic heart tissue only when reperfusion started.
Apstatin has excellent pharmacological properties and exhibits reasonable potency (micromolar), specificity and metabolic stability. However, it has chemical properties that limit its usefulness as an orally active drug. Although apstatin and related compounds have potential as injectable drugs, the potency and predicted intestinal absorption rate are probably too low to allow them to be effective following oral administration. Oral bioavailability is an essential property when the drug is being used in a chronic situation, such as adjunct treatment for hypertension or chronic heart or kidney disease. Therefore, it is an object of the present invention to discover APP2 inhibitors having greater potency (i.e., a lower IC50) than apstatin such that they can be administered in lower dosages as injectable drugs, and/or that because of their potency and chemical structure can be administered in an orally acceptable form.
A novel compound for increasing bradykinin levels is an α-hydroxy, β-amino tripeptide to decapeptide analog, which is disclosed in U.S. Pat. No. 5,656,603 to William H. Simmons, entitled “Aminopeptidase P inhibitors and Uses Thereof.”
Another novel compound for increasing bradykinin levels is disclosed in U.S. Pat. No. 7,390,789, entitled “Thio-Containing Inhibitors Of Aminopeptidase P, And Compositions Thereof,” which issued to William Simmons on Jun. 24, 2008, and is incorporated herein by reference for its disclosures relating to APP2 (mAPP therein) and ACE inhibitors. The '789 patent discloses various thio-containing tripeptide analogs that in the concentration range of 10 to 3400 nM inhibit purified rat membrane aminopeptidase P by 50% using Arg-Pro-Pro (0.5 mM) as the substrate. However, when these compounds were optimized for metabolic stability and desirable pharmacokinetic properties, they exhibited a loss of target specificity. They also inhibited ACE at concentrations close to the blood levels required to produce a sustained, complete inhibition of APP2.
Current treatment guidelines for acute myocardial infarction state that ACE inhibitors should not be given to patients at the time of reperfusion because of their ability to rapidly decrease blood pressure, primarily by blocking angiotensin II formation. On the other hand, a specific APP2 inhibitor by itself does not decrease blood pressure. Therefore, a compound that inhibits APP2 but not ACE should be safer to use at the time of reperfusion for enhancing the cardioprotective effects of endogenous bradykinin. Accordingly, an object of the present invention is to find a compound that greatly inhibits APP2 while minimally inhibiting ACE